Omar Calderon Jr, Marquette University

Prion diseases are neurodegenerative disorders that affect humans and animals and are characterized by long incubation periods, sponge like appearance in brain structure, neuronal loss, and inflammatory response failure. A prion is a spontaneously misfolded protein that is able to propagate by converting normally folded versions of itself to the misfolded form. Accumulation of a misfolded protein is associated with cell death and over the course of years may lead to a human brain filled with “holes.” In order to facilitate our understanding of prion disease, much work has been performed in the yeast model system. Several prions exist in yeast that have similarities to mammalian prions such as protein misfolding and infectivity. A model organism such as yeast can replicate quickly, be manipulated easily, and diminishes the risk of prion infectivity. The most studied yeast prion is [PSI+], which is the misfolded form of Sup35. Studies of [PSI+] have shown that prion appearance occurs through multiple steps and that several gene deletions diminish prion induction in different stages of the cycle. BEM1 has previously shown to decrease prion induction nearly 15 fold and is the focus of this project. Bem1p is a scaffolding protein that binds Cdc24p and Cdc42p to initiate actin polymerization. We hypothesized that the actin networks established by these protein interactions are required for prion appearance. Using a reverse genetics approach, point mutations in BEM1 at the sites of Cdc42p and Cdc24p interactions are being tested to understand how these protein interactions play a role in prion appearance.

Kaitlyn Curtis, Lewis University

The microbial community in penguin guano and its potential for methane production

Recently, the importance of an organism’s gut microbiome to its wellbeing has become apparent. One group of species we are interested in are penguins, who represent a major connection between terrestrial and aquatic environments. Much is still unknown about the penguin gut microbial community. One mystery associated with it has to do with penguin guano resting on mineral soil (i.e., ornithogenic soli). One group of scientists found penguin ornithogentic soil in Antarctica was a source of methane, assumed to be from a group of Archaea known as methanogens, the primary biological producers of methane. Other researchers could not detect the presence of methanogens in ornithogenic soil. Methanogens are normal members of the gut microbiota in many animals, including sea birds. Thus, we hypothesized that methanogens were the source of the methane reported from penguin guano. We tested this by extracting DNA from captive penguin fecal material and PCR and qPCR amplification of methanogen specific 16S rRNA and mcrA (a functional gene specific for methanogens) genes. All analyses showed that methanogens were below detection. Moreover, to understand the relationships between the gut microbes of captive penguins, a fingerprint of their bacterial community was analyzed, after 16S rRNA gene amplification, using denaturing gradient gel electrophoresis (DGGE). The DGGE showed differences between species, but a similarity between members of the same species. The question of who is responsible for the methane remains unanswered. The key to unraveling this mystery will be to determine the mechanism responsible for methane production from penguin guano.

Bryan Eder, Coe College

Nuclear RNA surveillance/degradation (NRS) is an essential process for maintaining cellular homeostasis. The RNA exosome is one of the main complexes responsible for RNA degradation, but the exosome does not degrade its substrates efficiently without cofactors, one of which is the TRAMP complex. TRAMP is comprised of three proteins that are integral to the identification and adenylation of RNA substrates for processing or degradation. Mtr4, a subunit of TRAMP, is an RNA helicase that serves to restructure the RNA so the RNA exosome can recognize and degrade it. The focus of this research is to determine what genetic elements control expression of the mouse homolog of mtr4, skiv2l2. Previous studies have shown induction of N2A differentiation into neurons results in downregulation of skiv2l2, and conversely, that knockdown of skiv2l2 results in greater N2A differentiation to neurons. These findings suggest that skiv2l2 is important to maintaining the proliferative state in the N2A cell line. Because gene expression is a reflection of both transcription and degradation of mRNA, this study sought to determine whether the 3’ untranslated region of skiv2l2 has an effect on skiv2l2 expression in N2A cells. First, the 3'UTR of skiv2l2 was amplified from N2A genomic DNA using high-fidelity PCR and cloned into a dual-luciferase vector, pmirGLO. Upon transfection of N2A cells with both this construct and unmodified pmirGLO vector, luciferase expression was quantified. This study found that the 3’ UTR of skiv2l2 downregulates luciferase expression, suggesting that this region possesses a controlling element that regulates skiv2l2 expression.

Chandler Farris, Marquette University

Proteins play an essential role in cellular structure, growth, and replication. A cell’s inability to regulate the disposal of misfolded proteins can cause different diseases. An example of a protein misfolding disorder is Prion disease. Prions are proteins that spontaneously misfold and recruit proteins of the same kind into their nonfunctional conformer. [PSI+] is a self-propagating prion form of the termination factor Sup35p and is the most well understood yeast prion. In yeast, prion formation involves multiple steps that ultimately lead to transmission of prion particles into daughter cells. It is suspected that the prion particles take advantage of actin tracts and move into the bud site during cell division. Previous work identified six gene deletions that reduce prion formation, most of which were associated with actin polymerization. Therefore, it is hypothesized that normal actin polymerization dynamics are necessary for prion formation. To test our hypothesis, we used two cytoskeletal drugs that interfere with both ends of F-actin polymers, latrunculin A (polymerization regulation) and cytochalasin D (depolymerization inhibitor). With the use of a prion-inducing plasmid, the prion domain of Sup35p was tagged with green fluorescent protein and overexpressed in yeast cells. Yeast cells that were treated with the drugs were viewed under the microscope to track two [PSI+] phenotypes, rings and aggregates. Preliminary data showed a decrease in the appearance of these phenotypes as the concentration of each drug was increased. Population samples showed a range of 3-8% drop in ring and aggregate appearance after drug treatments. This data suggests that actin’s role in prion induction is not limited to filamentous growth, and that depolymerization is also important. These findings will be confirmed by additional trials in the forthcoming weeks. Future experiments will be addressing how a specific actin polymerization inhibitor (jasplankinolide) affects prion appearance.

John Galatowitsch, Marquette University

Pyruvate carboxylase (PC) is allosterically regulated to a high degree, and this allosteric regulation has a major impact on cellular metabolic functions that influence cell proliferation and glucose mediated insulin release. Both A. nidulans PC and R. etli PC are allosterically inhibited by L-aspartate, but A. nidulans PC is inhibited to a greater degree. The binding site of L-aspartate is unknown, and it is unclear whether the binding site is conserved between species. To investigate the locus of action of L-aspartate inhibition, chimeric enzymes were created. These chimeras split a known regulatory binding site in two, with one half coming from R. etli PC and the other from A. nidulans PC. While both chimeric enzymes were active, they exhibited substantially reduced activity as compared to the wild-type enzymes. As expected, L-aspartate inhibits both wild-type enzymes. However, it had differing effects on the chimeras. One chimera displayed increased inhibition over the wild-types while the other was surprisingly activated by L-aspartate. These results provide a clearer description of where L-aspartate is mediating its allosteric effects, and can be used to further elucidate the binding site and mechanism of this regulation.

Jaime N. Lisack, Ithaca College

Homeostasis is required for cells to function, and if it fails the organism is likely to die due to numerous defects. With environments that constantly change in temperature, mechanisms have arisen to aid maintenance of homeostasis. An example of this is the heat shock response. At high temperatures, proteins fail to fold properly, resulting in malfunction and eventual cell death. The heat shock response triggers the creation of molecular chaperones called heat shock proteins (HSPs) to help proteins fold correctly. We are studying this highly conserved stress response in C. elegans. Although much is known about the heat shock response at high temperatures (30°C-33°C), little is known about heat shock at mild temperatures (26°C-27°C). This project explored HSP expression and effects of overexpression of HSP90 in mutants that fail to develop at mild heat shock. To examine changes in gene expression, changes in GFP expression from transgenes using HSP promoters were measured. It was found that HSP expression is evident at 26°C, increases at 27°C, and moves from head to tail over time. Finally, consequences of HSP expression in relation to other gene products was studied. This was done by overexpressing HSP90 in mutant C. elegans that arrest at mild temperatures due to over expression of proteins in their intestines. The overexpression of HSPs was unable to rescue this high temperature arrest phenotype. It appears HSPs play an active role in cell life at mild temperatures but the extent of which has not been fully uncovered.

TeNeal Metcalf, Tarrant County College

Two approaches for quantitative analysis of antibiotic resistance in the microbiome of invasive mussels

The resistome is the sum total of antibiotic resistance genes (ARG) present in the environment. Resistance determinants occur naturally and may spread through the bacterial community via horizontal gene transfer resulting in abundance of antibiotic resistant bacteria (ARB). The objective of this study was to determine whether ARG and ARB populations in the Lake Michigan ecosystem resistome are correlated with levels of anthropogenic pollution. The aquatic resistome was studied through sampling of the invasive mussel microbiome; this approach was evaluated using quantitative real-time polymerase chain reaction (qPCR) of ARG and culturing on media containing antibiotic. Field specimens of quagga (Dreissena bugensis) mussels collected at the Milwaukee Harbor were exposed to tetracycline at levels of 0.1, 1, and 10 ng/mL, and sampled for 120 hours. After depuration, mussels were exposed to lake water post sewage outflow, and sampled every 24 hours. Mussel tissue from tetracycline exposure was cultured to R2A media to determine heterotrophic community abundance, and to media containing antibiotics to determine the percentage of the community expressing clinical resistance. Additionally, bacterial abundance during exposures were quantified with qPCR using bacterial universal primers for 16s rRNA .16s rRNA detected during contaminated lake water exposure increased from 10x9 copy numbers to 10x11 during the observed uptake cycle, and returned to baseline during depuration. ARG were quantified based on tetW presence, which was detected at levels of 10x6 copies per gram tissue at baseline with an increase of two log orders during contaminated lake water exposure.

Elisia Meyle, Marquette University

Pyruvate Carboxylase (PC) is a biotin-dependent enzyme which catalyzes the formation of oxaloacetate to replenish crucial intermediates of the citric acid cycle. The activity of PC is contingent on the ability of the biotin cofactor to transport carbon dioxide from the biotin carboxylation domain to the carboxyl transferase domain. Computational studies suggest that the carboxybiotin intermediate has a unique effect on the enzyme conformation. However, there are no structures available detailing the binding of carboxybiotin in the enzyme active sites. This study aims to investigate the effect of the carboxylated cofactor on the structure and activity of PC by using stable acetyl biotin maleimides to serve as carboxybiotin analogues. A commercially available biotin maleimide analogue was selectively attached to Rhizobium etli. K1119C PC and this attachment was confirmed using streptavidin conjugated Qdot in a western blot protocol. Although the commercially available biotin maleimide is bound specifically to K1119C PC, kinetic studies indicated that it was unable to restore enzyme activity. Because of this limitation of the commercially available analog, synthesis of novel analogues with a variety of linker lengths was undertaken. A crystal structures using the acetylated form of this analogues will ultimately provide valuable insight regarding the effect that carboxybiotin has on the conformation of PC.

Kylli Paavola, Marquette University

Conjugative plasmids are contributing to the resistome of quagga mussels

Antibiotic resistant bacteria (ARB) and genes (ARG) occur naturally in the environment and their presence can be increased by antibiotic pollution. Invasive mussel species that reside in the Milwaukee harbor receive this pollution resulting in the accumulation and release of ARB and conjugative plasmids from their gut. A laboratory feeding experiment was designed to show the uptake and depuration rate of E. coli CV601gfp into the gut of the mussel. Plating on TSA plates with Kanamycin (Km, 100 ng/uL), Rifampicin (Rif, 50ng/uL), and Cyclohexamide (CXM,100 ng/uL) allowed for the strict isolation of CV601gfp, confirmed by UV. Utilizing this gfp-labeled strain that has no native plasmids and is not resistant to the antibiotics tested allowed for the screening of ARG-carrying plasmids from the mussels microbiome. We found that the gut of the filter feeding mussel harbor bacteria with conjugative plasmids, and it is also a natural environment that promotes horizontal gene transfer even with the rapid intake and depuration rates. In addition, conjugation experiments with E. coli CV601gfp and mussel tissue bacteria, from mussels collected from Milwaukee Harbor and Little Cedar Lake, were conducted to test for presence of plasmids with ARG. The conjugation mix was plated on TSA plates with Km/Rif/CXM and six different antibiotics. Mussels from the less polluted Little Cedar Lake showed resistance to less antibiotics, than the mussels from the Milwaukee harbor, most likely due to different levels of lake pollution and human impact.

Kevin G. Sanchez, Marquette University

Changes in temperature affect the basic physiological and molecular functions of organisms. In the Petrella Lab, we investigate how changes in temperature affect the fertility of the various Caenorhabditis species. Our research so far has occurred under standard lab conditions, where nematodes are placed on agar plates with an abundant food source and no risk of predation. Such an environment is beneficial to the survival and reproduction of Caenorhabditis nematodes. However, since this is unlikely to reflect conditions worms encounter in the wild, we cannot assume that how Caenorhabditis nematode responds in the lab is necessarily how it responds in an ecologically relevant setting. This summer, we did several things to try and overcome this issue. First, we isolated 19 nematodes from the wild, taking note of important ecological factors. Then, we identified and characterized these nematodes using molecular and morphological techniques. As these worms have not adapted to lab conditions as existing strains of Caenorhabditis likely has over the years, they will provide a better perspective of how nematodes react in the wild. Finally, we established microcosms, mini-habitats that contain moist soil and a food source, in order to mimic the nematodes natural habitat. In these microcosms we constructed ecologically relevant environments in which nematodes survived and reproduced for up to 14 days. We now can use these microcosms to model how populations of nematodes in the wild respond to changes in temperature, which brings new and exciting fields of study to this area of research.

Danielle Scheunemann, Robert Morris University

In Drosophila, tdc1 is the gene that encodes for tyrosine decarboxylase, the enzyme that converts tyrosine into tyramine. tdc1 homozygous mutants are sterile due to the shutdown of oogenesis which may suggest tyrosine has a role in its regulation. Based on studies in other organisms, we hypothesize that higher tyrosine levels may lead to activation of AMPK which then causes sterility. In order to investigate the morphology of tdc1 mutant egg chambers, nurse cell (NC) nuclei were stained with DAPI and follicle cells (FC) were stained with Fasciclin III. tdc1 mutants had degenerating NC nuclei in most egg chambers, but the FCs were intact indicating that shutdown of oogenesis is similar to a starvation induced shutdown. Caspase staining was done and reaffirmed that there was apoptosis in the mutant egg chambers. Because the tdc1 mutant phenotype resembles starved flies it was important to quantify the expression of tdc1 and the tyrosine metabolizing enzyme tyrosine aminotransferase gene via qPCR in starved wildtype flies. There were no consistent changes in the expression of tdc1, or tyrosine aminotransferase. Expression of AMPK in the FCs was altered by overexpressing AMPKdead mutants and AMPKconstitutively active mutants with the CY2gal4 driver. Egg chamber counts showed that the AMPKconstitutively active mutants had fewer vitellogenic egg chambers than AMPKdead mutants. Although the AMPKconstitutively active mutants did not have zero vitellogenic egg chambers like tdc1 mutants, this indicates that higher levels of AMPK could lead to sterility.

Ashtyn T. Smith, Westminster College

GABA, an inhibitory neurotransmitter, interacts with two receptors in the brain, GABAAR and GABABR. In adults, the ionotropic receptor (GABAA) is targeted with anti-seizure medication to manage epilepsy and similar disorders. This treatment is less effective in infants, however, due to the excitatory behavior exhibited by GABA during early brain development. For this reason, research has turned to the metabotropic receptor, GABABR, as a potential pharmacological target in epileptic neonates. Within neonatal hippocampal neurons, activation of GABAB receptors enhances current through voltage-gated L-type calcium channels (LTCC). It is uncertain whether one or both LTCC isoforms, CaV1.2 and CaV1.3, are modulated by GABABR. The Mynlieff lab previously demonstrated high expression of CaV1.2 during the early neonatal period as well as colocalization with GABABR in the superior region of the hippocampus. The present study sought to determine the feasibility of examining GABABR-mediated current enhancement through morpholino oligonucleotide knockdown of LTCCs in cultured hippocampal neurons and ratiometric calcium imaging. Cultured hippocampal neurons from postnatal 6-8 day old rats were treated with morpholinos designed against the mRNA of CaV1.2 for 24-48 hours. Calcium influx across voltage-gated channels was monitored during cell depolarization induced with high KCl solution. Significant differences in intracellular calcium were not detected between control cultures treated with non-sense morpholinos and cultures treated with CaV1.2 morpholinos (N = 137, 70). These results suggest insufficient knockdown of CaV1.2, or that CaV1.2 does not contribute enough current to the total calcium influx to be detected by this method.

Jordan Smith, Baldwin Wallace University

Reduced body mass demonstrated in drop-dead mutant larvae is not associated with hypoxia

Larval development plays a major role in determining adult body size in insects. Mutations in the drop dead (drd) gene, found in Drosophila melanogaster, cause abnormal phenotypes in the adult fly, such as tracheal defects and reduced body mass. Although the structure of the respiratory system is affected in adult drd mutant flies, these flies do not appear to be hypoxic. Larval hypoxia causes a reduced body mass similar to the drd mutants. Larvae were used to examine the relationship between reduced body mass and hypoxia. White pre-pupae were sorted and weighed by genotype and sex. Female mutant white pre-pupae showed a 20-25% decrease in body mass, but no such difference was seen in males. These results indicate that mutant females develop abnormally. The absence of a difference in males could be due to the balancer chromosome in “wildtype” males. To determine a potential role of drd in the tracheal system, adults in which drd was knocked down in the tracheae were collected and weighed. Male and female knockdowns showed an 18-25% decrease in body mass. Morphology of the tracheal system was also examined in drd mutant and wildtype larvae. Previous studies have shown that hypoxic flies have gnarled and disorganized tracheal branching. However, drd larvae did not show this phenotype. Real time qPCR determined that expression of the hypoxia-induced gene tango was not significantly upregulated in the drd mutants. Overall, the data suggest that drd mutants are not hypoxic and their reduced body mass must have another biological cause.

Megan Taylor, Manchester University

Identification and functional analysis of cold tolerance genes and mechanisms in Oryza sativa

Commonly referred to as Rice, Oryza sativa L. is a cereal crop from the plant family Poaceae, which is the grass family. Rice, in general, contributes 21% of the world’s calorific needs and 76% of Asia’s calorific needs. The importance of rice as a cereal grain is increasing as gluten intolerance and the incidence of celiac disease rises. This project began with phenotypic determination, DNA extraction/isolation, molecular genotyping, and utilization of genetic mapping software (Cartographer) for 93 backcross lines (BC1F2 plants, including 3 parental controls). The backcross involved Carolino 164, a cold-sensitive aus O. sativa ssp. from Chad (line 1654), and Wir 911, a cold-tolerant japonica O. sativa ssp. from Russia (line 1685). The F1 hybrid was backcrossed with the cold-sensitive 1654 parent and BC1F2 seeds were obtained from 93 BC1 lines. Abiotic environmental stressors such as low temperatures can induce oxidative stresses upon plants that can ultimately lead to death. Survival was measured for 15 BC1F2 plants from each of the 93 BC1 lines (completed in quadruplets) under severe cold temperature stress. PCR marker based quantitative trait loci (QTL) mapping was performed for the following traits: low temperature survival at the seedling stage (LTSS), plumule growth after cold stress (PGC), electrolyte leakage, and seed weight. Further, biochemical assays, such as electrolyte leakage, total chlorophyll content, catalase activity, and antioxidant activities were completed to uncover the mechanism, which cold tolerance could be controlled. High electrolyte leakage was correlated to low survival among the rice plants used in this study and suggests that membrane damage is the primary cause of death for cold sensitive rice varieties.

Daniel VanDerhoef, Marquette University

Colocalization of interneuron markers with the GABAB receptor in the CA1 region of the rat neonatal hippocampus

Intracellular calcium is a critical component in many cellular processes, especially within neuronal function. The Mynlieff laboratory has focused on modulation of voltage-dependent calcium channels mediated by the G-protein coupled GABAB receptor (GABABR). When GABAB receptors are activated, L-type current enhancement is seen only within 10-20% of neurons cultured from the superior region of the hippocampus. The Mynlieff laboratory has previously demonstrated that the calcium current enhancement is mediated through the Gq isoform of GABABR and subsequent activation of PKCα, but the specific L-type channel isoform modulated by the GABAB receptor is currently unknown, as well as the cell type in which this enhancement occurs. The goal of the present study was to determine the colocalization of components of the GABAB signaling pathway with neurochemical markers specific to interneuron subtypes to determine the cell type in which calcium current enhancement occurs. Cross sections of hippocampi from postnatal day 6-8 Sprague Dawley rat pups were imaged with a confocal microscope. This project sought to colocalize GABABR with cholecystokinin (CCK), calbindin, and neuropeptide Y (NPY) all of which have been previously identified as key indicators of interneuron subtypes and have colocalized with GABABR. In the superior region of the hippocampus, 1.64% of all neurons contain both calbindin and GABABR, 0.362% of all neurons contain CCK and GABABR, and 5.02% of all neurons contain NPY and GABABR. Considering that 10-20% of neurons in culture demonstrated the GABABR mediated response, it may be a combination of several interneuron subtypes that contribute to this enhancement.

Christina Wenn, Saint Mary’s College

The biflagellate green alga, Chlamydomonas, is a powerful model organism for the study of flagella, the dual signaling center and motile device for eukaryotes. Flagella are supported by extraordinarily stable microtubules that are nucleated from a basal body. In addition, numerous microtubules also radiate from the basal body region. Flagella amputation will lead to rapid assembly of new flagella. How cells redeploy tubulins for generation of flagella remains unknown. To explore this, we expressed, as a microtubule-dynamic reporter, a fluorescent protein-tagged EB1. EB1 is a plus-end tracking protein that binds the plus end of only growing microtubules, and is enriched at the tip of full length flagella. We generated transgenic strains that express either EB1-GFP or EB1-NeonGreen at the endogenous EB1 level for live cell imaging. We found that both molecules decorated flagellar tips, but EB1-NeonGreen was clearly superior. Interestingly, despite intense auto fluorescence from chlorophyll, EB1-NeonGreen decorated two types of microtubules differentially in the cell body: dynamic microtubules and stable microtubules. As expected, EB1-Neongreen appeared as a comet at the tips of numerous growing microtubules. Additionally, EB1-Neongreen decorated entire stable microtubule bundles, including the signature microtubule rootlets emanating from basal bodies. Following pH shock and de-flagellation, the stable population disappeared, while the comet-like tips of growing microtubules remained. This study demonstrated the solution to overcome auto fluorescence, and provided the first in vivo evidence that EB1 is capable of binding throughout microtubules, in addition to the tip. Thus, the differential associations are regulated, via signaling triggered by de-flagellation.

Stephanie Yeager, Marquette University

Determining the contribution of actin networks to early and late stage [PSI+] prion appearance in Saccharomyces cerevisiae

Prions are infectious proteins that develop from the spontaneous misfolding of naturally occurring proteins. Prions are found in mammalian and lower eukaryote systems, including yeast. In both systems, prions self-propagate and form insoluble aggregates of misfolded proteins. These misfolded aggregates act to further convert normal proteins into the prion conformation. Appearance of [PSI+], one of the most well studied yeast prions, results from the aggregation of a misfolded translation termination protein, Sup35. When the Sup35 prion domain is fused to GFP and overexpressed, a multistep process is observed during prion induction. First is the formation of a cytoplasmic ring, which is considered the early step of prion appearance, followed by the transfer of infectious oligomeric prion particles to daughter cells, which represents late stage prion appearance. Previous research has shown a link between actin and prion appearance, yet little is known about how actin networks may be involved in the transfer of prion particles to daughters. To determine if actin networks affect early or late stage prion appearance we tested the act1R177A mutation of the ACT1 gene, which is responsible for actin formation in yeast. The actR177A mutation showed a 75 fold decrease in prion induction. In addition, since actR177A mutants showed a 11.2 fold reduction in ring formation it suggests that ACT1 may play an important role in forming rings and therefore is an early class gene.